MiR-98-5p plays suppressive effects on IL-1β-induced chondrocyte injury associated with osteoarthritis by targeting CASP3

Background This study aims to explore how miR-98-5p affects osteoarthritis, focusing on its role in chondrocyte inflammation, apoptosis, and extracellular matrix (ECM) degradation. Methods Quantitative real-time PCR was used to measure miR-98-5p and CASP3 mRNA levels in OA cartilage tissues and IL-1β-treated CHON-001 cells. We predicted miR-98-5p and CASP3 binding sites using TargetScan and confirmed them via luciferase reporter assays. Chondrocyte viability was analyzed using CCK-8 assays, while pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were quantified via ELISA. Caspase-3 activity was examined to assess apoptosis, and Western blotting was conducted for protein marker quantification. Results Our results showed lower miR-98-5p levels in both OA cartilage and IL-1β-stimulated cells. Increasing miR-98-5p resulted in reduced pro-inflammatory cytokines, decreased caspase-3 activity, and improved cell viability. Furthermore, miR-98-5p overexpression hindered IL-1β-induced ECM degradation, evident from the decline in MMP-13 and β-catenin levels, and an increase in COL2A1 expression. MiR-98-5p's impact on CASP3 mRNA directly influenced its expression. Mimicking miR-98-5p's effects, CASP3 knockdown also inhibited IL-1β-induced inflammation, apoptosis, and ECM degradation. In contrast, CASP3 overexpression negated the suppressive effects of miR-98-5p. Conclusions In conclusion, our data collectively suggest that miR-98-5p plays a protective role against IL-1β-induced damage in chondrocytes by targeting CASP3, highlighting its potential as a therapeutic target for OA.

disorders.MicroRNAs, small (~ 22 nucleotides) endogenous non-coding RNAs, regulate gene expression at transcriptional and post-transcriptional levels by binding to the 3′-untranslated regions (3′-UTRs) of target mRNAs [10].Notably, the dysregulation of several miRNAs has been linked to cartilage degradation and OA progression [11][12][13].For instance, miR-296-5p was found to promote chondrocyte proliferation and inhibit apoptosis and matrix-degrading enzyme expression in response to IL-1β by targeting TGF-β1 [14].MiR-9, upregulated in OA rat cartilage, was shown to combat OA by reducing ECM degradation [15].Additionally, elevated miR-203a levels in OA tissues and models have been suggested to contribute to cartilage degradation by targeting Smad3 [16].MiR-98-5p, known for its dysregulation in inflammatory diseases such as ulcerative colitis [17], acute myocardial infarction [18], and asthma [19], has also been implicated in various biological processes.For example, it was involved in the injury of ox-LDL-induced HUVEC cells as a downstream gene of circ-USP36 [20], protected against cerebral ischemia/reperfusion injury [21], and was shown to impact bone regeneration by affecting osteogenic differentiation and osteoblast growth [22].Intriguingly, Huang et al. [23] identified miR-98-5p as a key miRNA in OA progression through bioinformatics analysis.
Caspases, a family of cysteine proteases comprising 15 members, are critical in programmed cell death and inflammation [24].Of these, caspase-3 (CASP3) is a prominent executor in the apoptotic process, extensively researched in relation to cancer development and therapy [25][26][27].CASP3's role in OA pathogenesis is also well-documented.For instance, Zou et al. [28] demonstrated that genistein's anti-apoptotic effects on chondrocytes, including reduced CASP3 expression, contribute to decreased inflammation and lessened cartilage degradation in OA treatment.High glucose levels over long periods have been found to increase CASP3 expression, leading to chondrocyte apoptosis and cytoskeleton aggregation [29].Thomas et al. [30] also highlighted that apoptosis-induced chondrocyte death could impact cartilage metabolism, underlining its significance in OA pathogenesis.Our previous studies align with findings by Huang et al. [23], suggesting CASP3 as a potential target of miR-98-5p.This leads to the hypothesis that miR-98-5p may mitigate OA by modulating chondrocyte inflammation, apoptosis, and ECM degradation through targeting CASP3.
In this study, we measured the expression of miR-98-5p and CASP3 in OA patient cartilage samples.Using miRNA target prediction, we verified the link between miR-98-5p and CASP3 in the IL-1β-stimulated chondrocyte cell line CHON-001.Our functional experiments focused on whether miR-98-5p influences IL-1β-induced inflammation, apoptosis, and ECM degradation in CHON-001 cells by targeting CASP3.

Clinical specimen collection
The blood and cartilage tissues from the femoral condyle and tibial plateau were obtained from twenty patients undergo total knee replacement for end-stage knee OA (13 males and 7 females, with an average age of 51.3 years old).Normal human articular cartilage without arthritis was collected from the knee or hip joints from 20 patients with osteosarcoma or trauma who were undergoing surgery (12 males and 8 females, with an average age of 45.6 years old) as normal control.For the current study, we specifically selected participants who primarily exhibited medial knee OA, as indicated by a medial joint space width (JSW) narrower than the lateral JSW based on radiographic evaluations.This approach aligns with the guidelines recommended by the Osteoarthritis Research Society International [31].Blood samples at baseline knee OA were centrifuged to extract serum for ELISA assay.Simultaneously, knee cartilage tissues were immediately preserved in liquid nitrogen for subsequent analysis.Informed consent was secured from all participating patients.This study adhered to the Declaration of Helsinki guidelines and received approval from the Ethics Committee of The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine (Heilongjiang Province, China).
In accordance with the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, USA), CHON-001 cells were transfected with 40 nM oligos or 1 μg vectors for 48 h, followed by 24 h stimulation with 10 ng/mL IL-1β.

Enzyme-linked immunosorbent assay (ELISA) assay
The serum was diluted at a 1:100 ratio and the supernatant from CHON-001 cells at a 1:50 ratio for the assessment of inflammation.The levels of interleukins IL-1β and IL-6, along with TNF-α, were quantified using specific ELISA kits (RayBiotech, Peachtree Corners, GA, USA).Three biological replicates were performed for the statistical analysis.

Cell viability assay
We performed cell counting kit-8 (CCK-8) assay to analyze the chondrocyte viability.In brief, cells were seeded into 96-well plates at a density of 4,000 cells per well.After culture for 24, 48 and 72 h, we discarded the supernatant and added 10 µl of CCK-8 solution (Beyotime, Shanghai, China) to each well.Following 2 h incubation at 37 °C, the absorbance at 450 nm was measured with MultiMode Microplate Reader (Thermo Fisher Scientific).Three biological replicates were performed for the statistical analysis.

Caspase-3 activity assay
To evaluate cell apoptosis, the caspase-3 activity was determined using a caspase-3 colorimetric assay kit (Abcam, Cambridge, UK) according to the instructions of manufacturer.Using a microplate reader (BioTek, Winooski, VT, USA), we measured the optical density value at 400 nm and normalized relative caspase-3 activity to the control group.Three biological replicates were performed for the statistical analysis.

Western blot
The protein samples of CHON-001 cells were extracted by radio-immunoprecipitation assay buffer (RIPA, Beyotime, China) and concentration was examined by a BCA protein assay Kit (Beyotime).Equal amount of protein (30 μg) was isolated by 10% SDS-PAGE and transferred into PVDF membranes.After performing 2 h blocking with 5% non-fat milk at room temperature, the membranes were probed with primary antibodies against caspase-3, Bcl-2, Bax, COL2A1, MMP-13, β-catenin and GAPDH (Abcam Cambridge, MA, USA) overnight at 4 °C.Subsequently, the membranes were incubated with the secondary antibody conjugated by horseradish peroxidase for 2 h at room temperature and then examined using enhanced chemiluminescence solution (Bio-Rad, Hercules, USA).Three biological replicates were performed for the statistical analysis.

Target prediction and luciferase reporter assay
The potential interaction between miR-98-5p and CASP3 was analyzed using TargetScan 7.1 (http:// www.targe tscan.org/).For the luciferase reporter assay, we created both wild-type (WT) and mutant (MUT) CASP3 luciferase reporter vectors.This involved inserting CASP3 3′-UTR sequences with either miR-98-5p binding or mutant sites into pmirGLO vectors (Promega, Madison, WI, USA).We then co-transfected CHON-001 cells with 0.2 µg of either WT or MUT CASP3 vectors and 20 nM miR-98-5p mimics or miR-NC.The cells were plated in 24-well plates at a density of 4.0 × 10 5 cells per well and transfected using Lipofectamine 2000 (Invitrogen).After 48 h incubation, we measured Firefly and Renilla luciferase activities using a Dual Luciferase Reporter Assay Kit (Promega), and the Firefly/Renilla luciferase ratio was used to determine relative luciferase activity.Three biological replicates were performed for the statistical analysis.

Statistical analysis
Data processing was performing with GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).All data were presented as the mean ± standard deviation (SD) from three independent biological replications.Statistical differences between two groups were assessed by Student's t-test and that among multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by Turkey's post hoc test.All p-values less than 0.05 were deemed statistically significant.

Decreased miR-98-5p and increased CASP3 in OA cartilage and cellular models
Initially, we gathered blood samples from 20 primary patients at baseline knee OA and 20 control subjects to assess inflammation levels.ELISA results indicated a substantial increase in pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the OA group compared to controls (Fig. 1A-C).We also measured miR-98-5p and CASP3 in OA cartilage tissues using quantitative real-time PCR. Figure 1D demonstrated a marked decrease in miR-98-5p levels in OA patient cartilage compared to controls.Figure 1E shows a significant increase in CASP3 mRNA in OA cartilage versus control tissues.Additionally, we used IL-1β to induce OA in CHON-001 cells in vitro.This resulted in a notable reduction in miR-98-5p and a rise in CASP3 mRNA, as illustrated in Fig. 1F.

Discussion
Growing research highlights the importance of miR-NAs in maintaining chondrocyte balance, a key factor in OA development [35].In our study, we noted a marked decrease in miR-98-5p and an increase in CASP3 in both OA cartilage and IL-1β-treated CHON-001 cells, relative to their respective controls.Functionally, miR-98-5p modulates CASP3, leading to reduced markers of inflammation, apoptosis, and extracellular matrix (ECM) degradation in these cells.These findings underscore miR-98-5p's potential role as a critical regulator in OA onset and progression, aligning with previously reported studies [23].

Conclusions
In conclusion, our research highlights the effects of miR-98-5p on alleviating IL-1β-induced inflammation, apoptosis, and ECM degradation in chondrocytes are mediated through regulation of CASP3.These insights present a promising target and lay a theoretical groundwork for future treatments of human OA.

Fig. 1
Fig. 1 Expression levels of miR-98-5p and CASP3 in OA cartilage and OA cellular model.(A-C) ELISA assay was applied to determine the levels of IL-1β, IL-6 and TNF-α in OA-affected blood samples (n = 20) and control samples (n = 20).Quantitative real time PCR was used to detect the expression levels of miR-98-5p (D) and CASP3 mRNA (E) in cartilage samples of OA patients (n = 20) and control samples (n = 20).**p < 0.01 and ***p < 0.001 indicate OA versus Control; (F) Quantitative real time PCR was used to detect the expression levels of miR-98-5p and CASP3 mRNA in IL-1β-induced CHON-001 cells and controls.***p < 0.001 indicates IL-1β versus Control